Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice

doi: 10.1007/978-1-4939-8831-0_8

Figure Lengend Snippet: Summary of mouse Cybb mutations from CRISPR targeting. Shown are the CRISPR target region and cut site (dotted line) for (a) exon 1 or (b) exon 3. Deletions matching MMEJ predictions are denoted by “*”. (c) Mutations matching predicted MMEJ-induced deletions. Wild-type sequences (bold) are listed in 5′ to 3′ orientation, with exons (uppercase), introns (lowercase), and CRISPR targets (underlined). CRISPR target and PAM sequences are shown in reverse complement, as they are on the complementary strand to the Cybb gene orientation

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 This Cybb knockout NSG mouse strain (NSG.Cybb[KO]), which contains a 235 bp deletion in Cybb exon 1, will be available from The Jackson Laboratory as JAX stock # 030610.

Techniques: CRISPR

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice

doi: 10.1007/978-1-4939-8831-0_8

Figure Lengend Snippet: Outlining of microinjection and embryo transfer procedures. Plugged female NSG mice are euthanized and fertilized eggs are collected from their oviducts. These zygotes are subsequently microinjected with Cas9 mRNA and either Cybb exon 1 or exon 3 sgRNA. After culturing injected eggs overnight, those that reach 2-cell stage of development are implanted into the oviducts of pseudopregnant surrogate mothers using a mouth-controlled glass transfer pipette

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 This Cybb knockout NSG mouse strain (NSG.Cybb[KO]), which contains a 235 bp deletion in Cybb exon 1, will be available from The Jackson Laboratory as JAX stock # 030610.

Techniques: Microinjection, Injection, Transferring

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice

doi: 10.1007/978-1-4939-8831-0_8

Figure Lengend Snippet: PCR and sequencing analysis of CRISPR-induced mutations in Cybb exon 1. (a) Gel electrophoresis of PCR products using primers 2 and 3 for mice (numbered 1–1 through 1–6), including water (H2O) and wild-type (wt) mouse DNA controls. (b) PCR analysis of mouse number 1–3 using primers 1 and 4 to amplify alleles with 235 and 240 bp deletions that appear as a single band. PCR analysis of this mouse using (c) primers 1 and 3 amplifies only the 235 bp deleted allele, while PCR using (d) primers 2 and 4 amplifies only the 240 bp deletion. (e) PCR genotyping of the Cybb exon 1 knockout mouse strain containing the 235 bp deletion established from mouse number 1–3, using primers 1 and 3 to distinguish between the 628 bp wild-type (wt) allele and the 393 bp knockout (ko) allele. (f) DNA sequences from PCR products for wild-type exon 1 and four mice with small deletions (at locations indicated by arrowheads)

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 This Cybb knockout NSG mouse strain (NSG.Cybb[KO]), which contains a 235 bp deletion in Cybb exon 1, will be available from The Jackson Laboratory as JAX stock # 030610.

Techniques: Sequencing, CRISPR, Nucleic Acid Electrophoresis, Knock-Out

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice

doi: 10.1007/978-1-4939-8831-0_8

Figure Lengend Snippet: Schematic interpretation of a prevalent microhomology-mediated end joining. Four out of seven offspring derived from embryos injected with Cybb exon 3 sgRNA carried exactly the same 3 bp deletion, supporting the notion that CRISPR-mediated deletions are not completely random. This diagram illustrates a plausible mechanism for interpreting biased deletion patterns mediated by short homologous sequences (in bold) near the two ends created by Cas9 cutting

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 This Cybb knockout NSG mouse strain (NSG.Cybb[KO]), which contains a 235 bp deletion in Cybb exon 1, will be available from The Jackson Laboratory as JAX stock # 030610.

Techniques: Derivative Assay, Injection, CRISPR